Abstract Cell-cell fusion is a cellular event that is critical to several developmental and physiological processes including bone homeostasis. Bone homeostasis is maintained by bone-forming osteoblasts and bone-resorbing osteoclasts. The fusion of osteoclasts is critical for osteoclast activation and function. Elevated osteoclastogenesis and bone resorption leads to osteoporosis that is a severe health problem characterized by imbalanced bone remodeling in aging and pathological conditions. Current anti-resorptive treatments for bone loss, which target early osteoclast differentiation and/or viability, have been limited by low response rates or side effects. Recent studies suggest that targeting molecules controling osteoclast fusion may be a better anti- resorptive therapeutic strategy for bone loss. However, very little is known about the molecular mechanisms underlying osteoclast fusion. We recently found that phospholipase D1 (PLD1), through generating the signaling phospholipid phosphatidic acid (PA), plays a critical role in osteoclast fusion. Our In vitro experiments show that PLD1 activity is activated during osteoclastogenesis. Inhibition or genetic deletion of PLD1 inhibit the fusion of mononucleated osteoclasts to multinucleated osteoclasts. PLD1 promotes osteoclast fusion through regulating the formation of the protrusive membrane structure at fusogenic synapses. Through both a PA- binding protein screen and RNA-Seq, we have identified a potential mechanism through which PLD1 regulates osteoclast fusion. Finally, global deletion of Pld1, the gene encoding PLD1, protects mice from bone loss. Based on these data, we hypothesize PLD1-generated PA plays a critical role in osteoclast fusion through regulating the formation of the protrusive fusogenic synapse. We will test our hypothesis with the following specific aims. In Aim 1, we will demonstrate that the spatiotemporal production of PLD1-genereated PA is critical for osteoclast fusion. In Aim 2, we will elucidate the mechanisms through which PLD1 coordinates actin cytoskeletal reorganization and membrane remodeling during osteoclast fusion. In Aim 3, we will define the role of osteoclast PLD1 in bone homeostasis in both physiological and pathological conditions using knockout mouse models. The goals of this project are to elucidate the role of PLD1 in osteoclast fusion and bone homeostasis and identify new and more effective therapeutic targets for osteoporosis and other bone diseases.